Pharmaceutical composition for inhibiting the growth of viruses and cancers

ABSTRACT

This invention is a method of treating cancer, both carcinomas and sarcomas, and viral infections, in particular HIV through the administration of a pharmaceutical composition containing a benzimidazole derivative. The composition is also claimed. The benzimidazole derivative is selected from the group consisting of:wherein X is hydrogen, halogen, alkyl of less than 7 carbon atoms or alkoxy of less than 7 carbon atoms; n is a positive integer of less than 4; Y is hydrogen, chlorine, nitro, methyl, ethyl or oxychloro; R is hydrogen, alkylaminocarbonyl wherein the alkyl group has from 3 to 6 carbon atoms, or an alkyl group of from 1 to 8 carbon atoms and R2 is 4-thiazolyl, NHCOOR1 wherein R1 is aliphatic hydrocarbon of less than 7 carbon atoms, prodrugs, pharmaceutically acceptable salts and mixtures thereof and a pharmaceutically acceptable carrier.

This is a continuation of application 10/106,429, filed Mar. 26, 2002,now U.S. Pat. No. 6,479,526 which is a continuation of U.S. Ser. No.09/748,651, filed Dec. 22, 2000, now U.S. Pat No. 6,362,207 issued Mar.26, 2002, which is a divisional application of U.S. Ser. No. 09/264,942,filed Mar. 9, 1999, now U.S. Pat. No. 6,262,093, which is acontinuation-in-part application of U.S. Ser. No. 08/927,550, filed Sep.6, 1997, now U.S. Pat. No. 5,880,144, which is a divisional of U.S. Ser.No. 08/771,193, filed Dec. 20, 1996, now U.S. Pat. No. 5,767,138, andwhich is a divisional of U.S. Ser. No. 08/420,914, filed Apr. 12, 1995,now abandoned. Application U.S. Ser. No. 09/264,942, now U.S. Pat. No.6,262,093 is also a continuation-in-part of U.S. Ser. No. 09/081,384,filed May 19, 1998, now abandoned, and also a continuation-in-part ofU.S. Ser. No. 09/081,627, filed May 19, 1998, now abandoned.

TECHNICAL FIELD

This invention is a method of treating cancer, both carcinomas andsarcomas, and viral infections, in particular HIV through theadministration of a pharmaceutical composition containing abenzimidazole derivative. The composition is also claimed.

BACKGROUND OF THE INVENTION

Cancers are the leading cause of death in animals and humans. The exactcause of cancer is not known, but links between certain activities suchas smoking or exposure to carcinogens and the incidence of certain typesof cancers and tumors has been shown by a number of researchers.

Many types of chemotherapeutic agents have been shown to be effectiveagainst cancers and tumor cells, but not all types of cancers and tumorsrespond to these agents. Unfortunately, many of these agents alsodestroy normal cells. The exact mechanism for the action of thesechemotherapeutic agents are not always known.

Despite advances in the field of cancer treatment the leading therapiesto date are surgery, radiation and chemotherapy. Chemotherapeuticapproaches are said to fight cancers that are metastasized or ones thatare particularly aggressive. Such cytocidal or cytostatic agents workbest on cancers with large growth factors, i.e., ones whose cells arerapidly dividing. To date, hormones, in particular estrogen,progesterone and testosterone, and some antibiotics produced by avariety of microbes, alkylating agents, and anti-metabolites form thebulk of therapies available to oncologists. Ideally cytotoxic agentsthat have specificity for cancer and tumor cells while not affectingnormal cells would be extremely desirable. Unfortunately, none have beenfound and instead agents which target especially rapidly dividing cells(both tumor and normal) have been used.

The development of materials that would target tumor cells due to someunique specificity for them would be a breakthrough. Alternatively,materials that were cytotoxic to tumor cells while exerting mild effectson normal cells would be desirable.

HIV and other viral infections are another leading cause of death. HIVis a disease in which a virus is replicated in the body which attacksthe body's immune system. The HIV virus is not easily destroyed nor isthere a good mechanism for keeping the host cells from replicating thevirus. Herpes Simplex is another viral infection which is difficult, ifnot impossible, to cure. A method of treating these diseases and otherviral infections is highly desirable. A material which would target theHIV virus and inhibit viral replication is highly desirable.

The benzimidazole derivatives used herein to treat cancer and/or viralinfection have been used as fungicides and as antihelmetics.

SUMMARY OF THE INVENTION

A method of treating cancer, in particular, treating cancers in warmblooded animals and humans, comprising administering a therapeuticallyeffective amount of a composition comprising a benzimidazole compoundselected from the group consisting of:

wherein X is hydrogen, halogen, alkyl of less than 7 carbon atoms oralkoxy of less than 7 carbon atoms; n is a positive integer of less than4; Y is hydrogen, chlorine, nitro, methyl, ethyl or oxychloro; R ishydrogen, alkylaminocarbonyl wherein the alkyl group has from 3 to 6carbon atoms, or an alkyl group of from 1 to 8 carbon atoms and R₂ is4-thiazolyl, NHCOOR₁ wherein R₁ is aliphatic hydrocarbon of less than 7carbon atoms, and preferably an alkyl group of less than 7 carbon atomsis claimed. Preferably the compositions contain:

wherein R is an alkyl of 1 through 8 carbon atoms and R₂ is selectedfrom the group consisting of 4-thiazolyl, NHCOOR₁, wherein R₁ is methyl,ethyl or isopropyl and the non-toxic, pharmaceutically acceptable acidsalts with both organic and inorganic acids. The most preferredcompounds are 2-(4-thiazolyl)benzimidazole,methyl-(butylcarbamoyl)-2-benzimidazolecarbamate and2-methoxycarbonylamino-benzimidazole and those wherein Y is chloro.

In the present invention it has been discovered that the compoundsdescribed above are useful for the inhibition of HIV and the treatmentof HIV infection and similar retrovirus infections. The presentinvention also provides methods for the treatment of HIV infectioncomprising administering to a host infected with HIV a pharmaceuticallyor therapeutically effective or acceptable amount of a compound asdescribed above, particularly those wherein R is 4-thiazolyl.

The present invention also provides a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a therapeuticallyeffective amount of a compound as described above.

These compositions have been discovered to inhibit the growth of canceror other tumors in humans or animals and to induce apoptosis of cancercells by administration of a therapeutically effective amount of thecomposition, preferably by administering a benzimidazole compound to thesite of the cancer.

More specifically, this invention provides an anti-cancer compositioncomprising a pharmaceutical carrier and a benzimidazole derivative asdefined herein along with a method for treating such cancers. Thesecompositions can induce apoptosis in cancer cells.

These compositions are also effective against viruses and are used totreat viral infections and this invention provides a method of treatingviral infections such as herpes, hepatitis, influenza and rhinoviruses.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

As used herein, a “pharmaceutically acceptable” component is one that issuitable for use with humans and/or animals without undue adverse sideeffects (such as toxicity, irritation, and allergic response)commensurate with a reasonable benefit/risk ratio.

As used herein, the term “safe and effective amount” refers to thequantity of a component which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of this invention.By “therapeutically effective amount” is meant an amount of a compoundof the present invention effective to yield the desired therapeuticresponse. For example to inhibit HIV infection or treat the symptoms ofinfection in a host or an amount effective to delay the growth of or tocause a cancer, either a sarcoma or lymphoma, to shrink. The specificsafe and effective amount or therapeutically effective amount will, varywith such factors as the particular condition being treated, thephysical condition of the patient, the type of mammal being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulations employed and the structure of thecompounds or its derivatives.

As used herein, a “pharmaceutical salts” is salt of the benzimidazolederivatives which are modified by making acid or base salts of thecompounds. Examples of pharmaceutically acceptable salts include, butare not limited to, mineral or organic acid salts of basic residues suchas amines; alkali or organic salts of acidic residues such as carboxylicacids. Preferably the salts are made using an organic or inorganic acid.These preferred acid salts are chlorides, bromides, sulfates, nitrates,phosphates, sulfonates, formates, tartrates, maleates, malates,citrates, benzoates, salicylates, ascorbates, and the like.

As used herein, a “pharmaceutical carrier” is a pharmaceuticallyacceptable solvent, suspending agent or vehicle for delivering thebenzimidazole derivatives to the animal or human. The carrier may beliquid or solid and is selected with the planned manner ofadministration in mind.

As used herein, “cancer” refers to all types of cancer or neoplasm ormalignant tumors found in mammals, including carcinomas and sarcomas.Examples of cancer are cancer of the brain, breast, cervix, colon, head& neck, kidney, lung, non-small cell lung, melanoma, mesothelioma,ovary, sarcoma, stomach, uterus and Medulloblastoma.

As used herein, the “benzimidazole derivatives” are the benzimidazoles,and their salts and also their prodrugs. The exact benzimidazoles aredescribed in detail below. The preferred materials are the products soldunder the names “Thiabendazole®”, “Benomyl®” and “Carbendazim®” by BASFand Hoechst, DuPont and MSD-AgVet.

As used herein “viruses” includes viruses which infect animals ormammals, including humans. Viruses includes retorviruses, HIV,influenza, polio viruses, herpes, herpes simplex, rhinoviruses,hepatitis, and the like.

B. The Benzimidazole Derivatives

The benzimidazole derivatives which are known for their antifungalactivities. They are systemic fungicides used to prevent and eradicatefungi. The compounds have the following structure:

wherein X is hydrogen, halogen, alkyl of less than 7 carbon atoms oralkoxy of less than 7 carbon atoms; n is a positive integer of less than4; Y is hydrogen, chlorine, nitro, methyl, ethyl or oxychloro; R ishydrogen, alkylaminocarbonyl wherein the alkyl group has from 3 to 6carbon atoms or an alkyl group having from 1 to 8 carbons, and R₂ is4-thiazolyl, NHCOOR₁ wherein R₁ is aliphatic hydrocarbon of less than 7carbon atoms, and preferably and alkyl group of less than 7 carbonatoms. Preferably the compositions are:

wherein R is an alkyl of 1 through 8 carbon atoms and R₂ is selectedfrom the group consisting of 4-thiazolyl, NHCOOR₁, wherein R₁ is methyl,ethyl or isopropyl and the non-toxic, pharmaceutically acceptable acidsalts with both organic and inorganic acids.

The most preferred compounds are 2-(4-thiazolyl)benzimidazole,methyl-(butylcarbamoyl)-2-benzimidazolecarbamate and2-methoxycarbonylamino-benzimidazole and the compounds wherein Y ischloro and X is hydrogen.

The benzimidazole compounds also include prodrugs. “Prodrugs” areconsidered to be any covalently bonded carriers which release the activeparent drug according to the formula of the benzimidazole derivativesdescribed above in vivo when such prodrug is administered to a mammaliansubject. Prodrugs of the benzimidazole compounds are prepared bymodifying functional groups present in the compounds in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compounds. Prodrugs include compounds whereinhydroxy, amine, or sulfhydryl groups are bonded to any group that, whenadministered to a mammalian subject, cleaves to form a free hydroxyl,amino, or sulfhydryl group, respectively. Examples of prodrugs include,but are not limited to, acetate, formate, or benzoate derivatives ofalcohol and amine functional groups in the benzimidazole derivatives;phosphate esters, dimethylglycine esters, aminoalkylbenzyl esters,aminoalkyl esters and carboxyalkyl esters of alcohol and phenolfunctional groups in the benzimidazole derivatives; and the like.

The pharmaceutically acceptable salts of the benzimidazole derivativesinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the benzimidazole derivatives formed, for example, fromnon-toxic inorganic or organic acids. For example, such conventionalnon-toxic salts include those derived from inorganic acids such ashydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric andthe like; and the salts prepared from organic acids such as acetic,propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric,ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic,benzoic, salicylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,and the like.

The pharmaceutically acceptable salts of the present invention aresynthesized from the benzimidazole derivatives which contain a basic oracidic moiety by conventional chemical methods. Generally, such saltsare prepared by reacting the free acid or base forms of these compoundswith a stoichiometric amount of the appropriate base or acid in water orin an organic solvent, or in a mixture of the two; generally, nonaqueousmedia like ether, ethyl acetate, ethanol, isopropanol, or acetonitrileare preferred. Lists of suitable salts are found in Remington'sPharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa.,1985, p. 1418, the disclosure of which is hereby incorporated byreference. The disclosures of all of the references cited herein arehereby incorporated herein by reference in their entirety.

Synthesis

The benzimidazole derivatives are prepared in a number of ways wellknown to one skilled in the art of organic synthesis. The benzimidazolederivatives are synthesized using the methods described below, togetherwith synthetic methods known in the art of synthetic organic chemistry,or variations thereon as appreciated by those skilled in the art.Preferred methods include but are not limited to those methods describedbelow. Each of the references cited below are hereby incorporated hereinby reference.

These compounds are prepared according to the method described in U.S.Pat. No. 3,738,995 issued to Adams et al, Jun. 12, 1973. The thiazolylderivatives are prepared according to the method described in Brown etal., J. Am. Chem. Soc., 83. 1764 (1961) and Grenda et al., J. Org.Chem., 30, 259 (1965).

C. Dosage and Dosage Delivery Forms

The type of compound and the carrier and the amount will vary widelydepending on the species of the warm blooded animal or human, bodyweight, and tumor being treated. The dosage administered will varydepending upon known factors, such as the pharmacodynamiccharacteristics of the particular agent and its mode and route ofadministration; the age, health and weight of the recipient; the natureand extent of the symptoms; the kind of concurrent treatment; thefrequency of treatment; and the effect desired.

The benzimidazole is preferably micronized or powdered so that it ismore easily dispersed and solubilized by the body. Processes forgrinding or pulverizing drugs are well known in the art. For example, ahammer mill or similar milling device are used. The preferred particlesize is less than about 100μ and preferably less than 50μ.

The dosage administered will vary depending upon known factors such asthe pharmacodynamic characteristics of the particular active ingredient,and its mode and route of administration; age, sex, health, metabolicrate, absorptive efficiency and/or weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency oftreatment; and the effect desired.

A “tumor growth inhibiting amount” of the benzimidazole derivatives isthat amount which is effective to inhibit or slow the growth of a tumor.

Dosage forms (compositions) suitable for internal administration containfrom about 1.0 milligram to about 5000 milligrams of active ingredientper unit. In these pharmaceutical compositions, the active ingredientwill ordinarily be present in an amount of about 0.5-95% by weight basedon the total weight of the composition. Based on the body weight of thepatent, the dosage may be administered in one or more doses severaltimes per day or per week. Multiple dosage units may be required toachieve a therapeutically effective amount. For example, if the dosageform is 1000 mg, and the patient weighs 40 kg, one pill will provide adose of 25 mg per kg for that patient. It will provide a dose of only12.5 mg/kg for a 80 kg patient.

The compounds have shown dose responsiveness in vivo against viruses andcancers in mice at 500 mg/kg, 2500 mg/kg, 3500 mg/kg, 4000 mg/kg, 5000mg/kg and 6000 mg/kg. Generally a dosage effective in mice translates toabout {fraction (1/12)} of the dosage required in humans. By way ofgeneral guidance, for humans a dosage of as little as about 30milligrams (mg) per kilogram (kg) of body weight and up to about 10000mg per kg of body weight is suitable. Preferably from 50 mg/kg to about5000 mg/kg of body weight is used. Most preferably the doses are between100 mg/kg to about 3000 mg/kg of body weight. However, a dosage ofbetween about 2 milligrams (mg) per kilogram (kg) of body weight toabout 400 mg per kg of body weight is also suitable for someindications.

Intravenously, the most preferred doses may range from about 1 to about1000 mg/kg/minute during a constant rate infusion. Benzimidazolederivatives may be administered in a single daily dose, or the totaldaily dosage may be administered in divided doses of two, three, or fourtimes daily. The benzimidazole derivatives are given in one or moredoses on a daily basis or from one to three times a week.

The benzimidazole derivatives may also be administered in intranasalform via topical use of suitable intranasal vehicles, or via transdermalroutes, using those forms of transdermal skin patches well known tothose of ordinary skill in that art. To be administered in the form of atransdermal delivery system, the dosage administration will, of course,be continuous rather than intermittent throughout the dosage regimen.

Generally, the dosage in man is lower than for small warm bloodedmammals such as mice. A dosage unit may comprise a single compound ormixtures thereof with other compounds or other cancer inhibitingcompounds or tumor growth inhibiting compounds or anti-viral compounds.The dosage unit can also comprise diluents, extenders, carriers and thelike. The unit may be in solid or gel form such as pills, tablets,capsules and the like or in liquid form suitable for oral, rectal,topical, intravenous injection or parenteral administration or injectioninto or around the tumor.

The benzimidazole derivatives are typically mixed with apharmaceutically acceptable carrier. This carrier can be a solid orliquid and the type is generally chosen based on the type ofadministration being used. The active agent can be coadministered in theform of a tablet or capsule, as an agglomerated powder or in a liquidform. Examples of suitable solid carriers include lactose, sucrose,gelatin and agar. Capsule or tablets are easily formulated and can bemade easy to swallow or chew; other solid forms include granules, andbulk powders. Tablets may contain suitable binders, lubricants,diluents, disintegrating agents, coloring agents, flavoring agents,flow-inducing agents, and melting agents. Examples of suitable liquiddosage forms include solutions or suspensions in water, pharmaceuticallyacceptable fats and oils, alcohols or other organic solvents, includingesters, emulsions, syrups or elixirs, suspensions, solutions and/orsuspensions reconstituted from non-effervescent granules andeffervescent preparations reconstituted from effervescent granules. Suchliquid dosage forms may contain, for example, suitable solvents,preservatives, emulsifying agents, suspending agents, diluents,sweeteners, thickeners, and melting agents. Oral dosage forms optionallycontain flavorants and coloring agents. Parenteral and intravenous formswould also include minerals and other materials to make them compatiblewith the type of injection or delivery system chosen.

D. Examples of Formulation

The benzimidazole derivatives of this invention are administered astreatment for cancer and viral infections, including retroviral, by anymeans that produces contact of the active agent with the agent's site ofaction in the body. The antitumor compounds (active ingredients) of thisinvention are administered to inhibit tumors by any means that producescontact of the active ingredient with the agent's site of action in thebody of a mammal. They can be administered by any conventional meansavailable for use in conjunction with pharmaceuticals, either asindividual therapeutic agents or in a combination of therapeutic agents.They can be administered alone, but generally are administered with apharmaceutical carrier selected on the basis of the chosen route ofadministration and standard pharmaceutical practice.

The benzimidazole derivatives are administered in oral dosage forms astablets, capsules (each of which includes sustained release or timedrelease formulations), pills, powders, granules, elixirs, tinctures,suspensions, syrups, and emulsions. The benzimidazole derivatives mayalso be administered in intravenous (bolus or infusion),intraperitoneal, subcutaneous, or intramuscular form, all using dosageforms well known to those of ordinary skill in the pharmaceutical arts.

In the methods of the present invention, the compounds herein describedin detail can form the active ingredient, and are typically administeredin admixture with suitable pharmaceutical diluents, excipients, orcarriers (collectively referred to herein as a pharmaceuticallyacceptable carrier or carrier materials) suitably selected with respectto the intended form of administration, that is, oral tablets, capsules,elixirs, syrups and the like, and consistent with conventionalpharmaceutical practices.

For instance, for oral administration in the dosage unit form of atablet or capsule, the active drug component can be combined with anoral, non-toxic, pharmaceutically acceptable, inert carrier such aslactose, starch, sucrose, glucose, methyl cellulose, magnesium stearate,dicalcium phosphate, calcium sulfate, mannitol, sorbitol and the like.

For oral administration in liquid dosage form, the oral drug componentsare combined with any oral, non-toxic, pharmaceutically acceptable inertcarrier such as ethanol, glycerol, water, and the like. Moreover, whendesired or necessary, suitable binders, lubricants, disintegratingagents, and coloring agents can also be incorporated into the mixture.Suitable binders include starch, gelatin, natural sugars such as glucoseor beta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth, or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes, and the like. Lubricants used in thesedosage forms include sodium oleate, sodium stearate, magnesium stearate,sodium benzoate, sodium acetate, sodium chloride, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthan gum, and the like.

The benzimidazole derivatives can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamallar vesicles, and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, such as cholesterol,stearylamine, or phosphatidylcholines.

Benzimidazole derivatives may also be coupled with soluble polymers astargetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxylpropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacylates, andcrosslinked or amphipathic block copolymers of hydrogels.

The active ingredient can be administered orally in solid dosage forms,such as capsules, tablets, and powders, or in liquid dosage forms, suchas elixirs, syrups, and suspensions. It can also be administeredparentally, in sterile liquid dosage forms.

Gelatin capsules may contain the active ingredient and powderedcarriers, such as lactose, starch, cellulose derivatives, magnesiumstearate, stearic acid, and the like. Similar diluents can be used tomake compressed tablets. Both tablets and capsules can be manufacturedas sustained release products to provide for continuous release ofmedication over a period of hours. Compressed tablets can be sugarcoated or film coated to mask any unpleasant taste and protect thetablet from the atmosphere, or enteric coated for selectivedisintegration in the gastrointestinal tract.

Liquid dosage forms for oral administration can contain coloring andflavoring to increase patient acceptance. In general, water, a suitableoil, saline, aqueous dextrose (glucose), and related sugar solutions andglycols such as propylene glycol or polyethylene glycols are suitablecarriers for parenteral solutions. Solutions for parenteraladministration preferably contain a water soluble salt of the activeingredient, suitable stabilizing agents, and if necessary, buffersubstances. Antioxidizing agents such as sodium bisulfite, sodiumsulfite, or ascorbic acid, either alone or combined, are suitablestabilizing agents. Also used are citric acid and its salts and sodiumEDTA. In addition, parenteral solutions can contain preservatives, suchas benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences, Mack Publishing Company, a standard referencetext in this field.

Useful pharmaceutical dosage forms for administration of the compoundsof this invention are illustrated as follows:

Capsules

A large number of unit capsules are prepared by filling standardtwo-piece hard gelatin capsules each with 100 to 500 milligrams ofpowdered active ingredient, 5-150 milligrams of lactose, 5-50 milligramsof cellulose, and 6 milligrams magnesium stearate.

Soft Gelatin Capsules

A mixture of active ingredient in a digestible oil such as soybean oil,cottonseed oil or olive oil is prepared and injected by means of apositive displacement pump into gelatin to form soft gelatin capsulescontaining 100-500 milligrams of the active ingredient. The capsules arewashed and dried.

Tablets

A large number of tablets are prepared by conventional procedures sothat the dosage unit was 100-500 milligrams of active ingredient, 0.2milligrams of colloidal silicon dioxide, 5 milligrams of magnesiumstearate, 50-275 milligrams of microcrystalline cellulose, 11 milligramsof starch and 98.8 milligrams of lactose. Appropriate coatings may beapplied to increase palatability or delay absorption.

Injectable

A parenteral composition suitable for administration by injection isprepared by stirring 1.5% by weight of active ingredient in 10% byvolume propylene glycol and water. The solution is made isotonic withsodium chloride and sterilized.

Suspension

An aqueous suspension is prepared for oral administration so that each 5ml contain 100 mg of finely divided active ingredient, 200 mg of sodiumcarboxym ethyl cellulose, 5 mg of sodium benzoate, 1.0 g of sorbitolsolution, U.S.P., and 0.025 ml of vanillin.

The present invention also includes pharmaceutical kits useful, forexample, for the treatment of HIV infection, which comprise one or morecontainers containing a pharmaceutical composition comprising atherapeutically effective amount of a benzimidazole derivative. Suchkits may further include, if desired, one or more of variousconventional pharmaceutical kit components, such as, for example,containers with one or more pharmaceutically acceptable carriers,additional containers, etc., as will be readily apparent to thoseskilled in the art. Printed instructions, either as inserts or aslabels, indicating quantities of the components to be administered,guidelines for administration, and/or guidelines for mixing thecomponents, may also be included in the kit. In the present disclosureit should be understood that the specified materials and conditions areimportant in practicing the invention but that unspecified materials andconditions are not excluded so long as they do not prevent the benefitsof the invention from being realized.

The following examples are illustrative and are not meant to be limitingto the invention.

Colon, Breast and Lung Tumor Cells Test

The following cell culture tests were performed to test the toxicity ofthe benzimidazole compounds on colon, breast and lung human tumor cells.The viability of the cells were tested by looking at MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)reduction. MTT assay is a well known measure of cell viability.

The colon tumor cells (HT29 from American Type Culture Collection (ATCC)and the breast cells (MX1 from cell lines from ATCC) were cultured inEagle's Miminal Essential Medium with 10% fetal bovine serum. The lungtumor cells (A549 from ATCC cell lines) were cultured in Ham's F12medium with 10% fetal bovine serum.

The tumor cells were passaged and seeded into culture flasks at thedesired cell densities. The culture medium was decanted and the cellsheets were washed twice with phosphate buffered saline (PBS). The cellswere trypsinized and triturated prior to seeding the flasks. Unlessotherwise indicated the cultures were incubated at 37±1° C. in ahumidified atmosphere of 5±1% carbon dioxide in air. The cultures wereincubated until they were 50-80% confluent.

The cells were subcultured when the flasks were subconfluent. The mediumwas aspirated from the flasks and the cell sheets rinsed twice with PBS.Next, the Trypsin Solution was added to each flask to cover the cellsheet. The Trypsin Solution was removed after 30-60 seconds and theflasks were incubated at room temperature for two to six minutes. When90% of the cells became dislodged, growth medium was added. The cellswere removed by trituration and transferred to a sterile centrifugetube. The concentration of cells in the suspension was determined, andan appropriate dilution was made to obtain a density of 5000 cells/ml.The cells were subcultured into the designated wells of the 96-wellbioassay plates (200 microliter cell suspension per well). PBS was addedto all the remaining wells to maintain humidity. The plates were thenincubated overnight before test article treatment.

Each dose of test article was tested by treating quadruplicate wells ofcultures with 100 microliter of each dilution. Those wells designated assolvent controls received an additional 100 microliter of methanolcontrol; negative controls wells received an additional 100 microlitersof treatment medium. PBS was added to the remaining wells not treatedwith test article or medium. The plates were then incubated forapproximately 5 days.

At the end of the 5 day incubation, each dose group was examinedmicroscopically to assess toxicity. A 0.5 mg/ml dilution of MTT was madein treatment medium, and the dilution was filtered through a 0,.45micrometer filter to remove undissolved crystals. The medium wasdecanted from the wells of the bioassy plates. Immediately thereafter,2000 microliter of the filtered MTT solution was added to all test wellsexcept for the two untreated blank test wells. The two blank wellsreceived 200 microliters of treatment medium. The plates were returnedto the incubator for about 3 hours. After incubation, the MTT containingmedium was decanted. Excess medium was added to each well and the plateswere shaken at room temperature for about 2 hours.

The absorbance at 550 nm (OD₅₅₀) of each well was measured with aMolecular Devices (Menlo Park, Calif.) VMax plate reader.

The mean OD₅₅₀ of the solvent control wells and that of each testarticle dilution, and that of each of the blank wells and the positivecontrol were calculated. The mean OD₅₅₀ of the blank wells wassubtracted from the mean of the solvent control wells, and test articlewells, respectively to give the corresponding mean OD₅₅₀₁.${\% \quad {of}\quad {Control}} = {\frac{{corrected}\quad {mean}\quad {OD}_{550}\quad {of}\quad {Test}\quad {Article}\quad {Dilution}}{{corrected}\quad {mean}\quad {of}\quad {OD}_{550}\quad {of}\quad {Solvent}\quad {Control}} \times 100}$

Dose response curves were prepared as semi-log plots with % of controlon the ordinate (linear) and the test article concentration on theabscissa (logarithmic). The EC₅₀ was interpolated from the plots foreach test article.

For the test articles administered in methanol, separate responses wereprepared to correct for the methanol data.3

Adriamycin was used as a positive control. In all cases, it was moretoxic than any of the test materials by one or two logs. Adriamycin isone of the more potent agents in current use and one with significantside effects. The peak plasma concentration of other, quite effectivechemotherapeutic agents may be 10 to 50 times higher than that ofAdriamycin.

The EC₅₀ is the concentration at which one half of the cells are killed.

TABLE 1 EC-50 Result (ppm) Test Material HT29 HT29 MX1 MX1 A549 A549Adriamycin 0.03 0.006 0.02 0.001 0.03 0.009 benomyl 0.742 0.747 1.422.42 0.980 1.02 carbendazim 0.621 0.662 0.829 0.856 0.856 0.836

In normal healthy cells, the following results were obtained. As isevident, the benomyl and carbendazim were much less toxic to normalhealthy cells than adriamycin.

TABLE 2 EC-50 Broncheal Kerotinoyle Test Material Cells CellsFibroblasts Benomyl 0.728 0.682 3.26 2.4 3.24 2.81 Carbendazim 0.3200.506 0.752 0.822 1.52 1.42 Adriamycin 0.015 0.0020 0.0035 0.0093 0.0650.10

In a related study using lung tumor cells (A-549) breast tumor cells(MCF-7) and colon tumor cells (HT-29), thiabendazole effectively killedthese cells. Table 3 summarizes the results

TABLE 3 Optical Density Concentration (ppm) A-549 MCF-7 HT-29 0-Control0.600 0.245 0.398 173 0.007 0.007 0.005  35 0.411 0.025 0.011  17.30.851 0.258 0.204  3.46 1.12 0.466 0.713  0.87 1.32 0.507 0.852

These experiments show that these compositions are effective in killingtumor cells of the breast, colon and lung type.

Carbendazim has shown broad-scale efficacy against multiple cancer typesboth in vitro and in vivo. The cancers tested include colon, lung,breast, prostate, pancreatic, leukemia, melanoma, neuroblastoma,ovarian, neck and head, and brain. Also multiple cell lines were testedin almost instances.

The initial efficacy is comparable to existing best available drugs. Butwith carbendazim, the tumors do not recur or reappear as happens withCytoxan and Taxol, which otherwise are quite good against breast cancer.Similarly, pancreatic cancer does not appear to come back as oftenhappens with Gemcitabine treatment.

Carbendazim is particularly good in mouse melanoma in mice, which manypeople believe is the best predictive model for efficacy in humans. Ithas shown outstanding broad and good results in the human tissue cloningtest. This is an in vitro test on conventionally treated and recentlyexcised human tumors.

Carbendazim is equally effective against p53 deficient/defective celllines, unlike most existing drugs. It appears that carbendazim inducesapoptosis in cancer cells at sub-lethal concentrations to normal cells.

Other benefits of carbendazim are:

Its oral LD₅₀ in mice is quite high (11,000 mg/kg), a low overalltoxicity unlike most cancer drugs. For perspective the LD₅₀ of tablesalt is 3750 mg/kg.

It is effective in cancers that form tumors and those that do not, e.g.both carcinomas and sarcomas.

The results of these studies are provided in more detail below.

Mechanism of Action Studies

Some of the pharmacological effects of carbendazim were demonstrated bystudying its ability to induce apoptosis in cancer cells, studying itseffect on p53-abnormal cell lines and determining during which cellularlife cycle phase carbendazim exerts its effects.

Apoptosis Study

Apoptosis is a specific type of cell death which differs from necrosisand is characterized by specific morphological, biochemical andmicellular cell changes. Abnormalities in p53 expression are generallylinked with the prevention of apoptosis and p53 abnormalities are commonin human tumors which are resistant to conventional cytotoxic agents.

Summary of Test Results

The extent of apoptosis is measured in human tumor cell lines aftertreatment for 1, 2, 3 and 4 days with carbendazim. At each of these timepoints, the cells are harvested and assayed using the terminaldeoxynucleotidyl transferase (TdT) assays. Both microscopy and flowcytometry were used for the TdT assay.

In MCF7, HT29, B16 and SK-MES cell lines, there was a concentrationdependent effect on cell growth. In most cases at concentrations ofcarbendazim greater than 1 μg/ml, the growth rate was significantlyslower than in the untreated samples. After completion of the studies onthe MCF7 and HT29 cell lines, it was evident that the 0.1 μg/mlconcentration had little effect on either apoptosis or cell growth.Therefore subsequent assays were with 5 μg/ml instead of 0.1 μg/ml.

The growth of MCF7 (breast cancer) cells was not significantly affectedby carbendazim below 10 μg/ml as shown by either method. However, at 10μg/ml the increase in apoptosis was evident at days 3 and 4. Theincrease in apoptosis was low, less than 10% at the high concentration.After normalization for the cell growth, carbendazim at 10 μg/ml had aneffect at day 4.

The growth of HT 29 (colon) cancer cells was not largely slowed down bycarbendazim below 10 μg/ml. Concentration-dependent increase inapoptosis was observed at days 3 and 4, reaching >25% apoptosis in thepresence of 10 ug/ml carbendazim. After normalization for the cellgrowth rates, the concentration-response effect was seen at day 4.

At all concentrations, carbendazim affected the growth rates in B16murine melanoma cell line. Some concentration dependent effects onapoptosis were seen at days 1-4 by TdT microscopy and days 2-4 by flowcytometry. The concentration effects at days 1-4 were much more evidentafter normalization of the apoptosis for the growth rates.

The growth of SK-MES cells was slowed done by all concentrations ofcarbendazim past day 1. The percent of apoptosis showed aconcentration-response effect at days 1-4 by microscopic TdT assay andby flow cytometry at days 3 and 4. A normalized graph showed aconcentration response effect of the compound on apoptosis at all days.

Conclusion

At concentrations less than 10 μg/ml, the greatest response tocarbendazim was seen in the SK-MES lines (lung), followed by B16 (murinemelanoma), HT29 (colon), and MCF7 (breast) cells. The HT29, SK-MES andB16 cell lines express abnormal p53. Accordingly carbendazim can induceapoptosis in p53 abnormal cell lines.

Selectivity in Killing p53 Abnormal Cell Lines

Carbendazim provides in vivo activity against HT29 tumor cells whichexpress abnormal p53.

Summary of test results

Pairs of tumors of the same type were chosen, one expressing normal p53and the other abnormal p53. Breast lines used were MCF7 for the normalp53, V4B, a MCF7 cells transformed with an empty vector, and VM4K, anMCF7 cells transfected with a vector encoding abnormal p53. In thecolorectal cancer model HCT116 with a normal p53 was used and DLD-1 wasused as the abnormal p53 cell line. The tumor cells were grown thepresence and absence of carbendazim for 7 days. Cell growth rates weredetermined in each group by counting the cell numbers daily using aCoulter counter.

In the breast model at day 7, using 1 μg/ml of carbendazim, cell countsin all three cell lines were between 50% and 60% of the control. Atconcentrations of 5 and 10 μg/ml the MCF7 cell counts were 45% and 36%of the control. The V4B and VM4K cell lines were less than 10% of therespective controls. The data from the VM4K cell line would alsoindicate the carbendazim is again selectively killing the p53 abnormalcells, however, there was also a marked decrease in the cell numbers inthe V4B line carrying only the empty vector. In light of the controversyregarding the p53 status in MCF7 cells, which some researchers claim toexpress abnormal p53, these differences may be more difficult tointerpret.

At day 7 in the colorectal model, at 1 μg/ml concentration carbendazimcell counts in the DLD1 line (abnormal p53) were only 34% of thecontrol. The HCT 116 cell line, (normal p53) showed cell counts thatwere 78% of the control suggesting that carbendazim may selectively killp53 abnormal cells. At carbendazim concentrations of 5 and 10 μg/ml,cell counts were less than 10% of the control in both the DLD-1 and HCT116 lines indicating that at the larger concentration, the drug wasequally toxic to cell lines containing abnormal and normal p53.

Conclusion

At higher concentrations, 5 and 10 μg/ml, the drug was equally toxic tocell lines containing abnormal p53 and normal p53 in both colon andbreast cancers.

In Vitro Studies of Carbendazim on Cancer Cell Lines

A dose response effect of carbendazim in a human tumor cloning formingunits study (HTCU) is summarized below. This is an in vitro test oftreatments on conventionally treated, then recently excised humantumors. It is an important study because carbendazim is showingeffectiveness against cell lines which have survived conventionaltreatment and which themselves have not had undergone too many passages(due to recent excision). This is significant since long living celllines undergo changes (passages), some of which may affect theirresistance to some drugs, and hemotherapeutic agent resistant cells canbe formed. The data show the activity as the tumors tested that had ≦50%survival (a high number is desirable).

In Vitro Human Tumor Colony Forming Units Test

Solid tumors removed by patients are minced into 2 to 5 mm fragments andimmediately placed in McCoy's Medium 5A plus 10% heat inactivatednewborn calf serum plus 1% penicillin/streptomycin. Within 4 hours,these solid tumors are mechanically disassociated with scissors, forcedthrough No. 100 stainless steel mesh, through 25 gauge needles, and thenwashed with McCoy's medium as described above. Ascitic, pleural,pericardial fluids and bone marrow are obtained by standard techniques.The fluid or marrow is placed in sterile containers containing 10 unitsof preservative free heparin per ml. of malignant fluid or marrow. Aftercentrifugation at 150×g for 10 minutes, the cells are harvested andwashed with McCoy's medium plus 10% heat inactivated calf serum. Theviability of cell suspensions is determined on a hemocytometer withtrypan blue.

Cells to be cloned are suspended in 0.3% agar in enriched CMRL1066supplemented with 15% heat inactivated horse serum, penicillin (100units/ml), streptomycin (2 mg/ml), glutamine (2 mM), insulin (3units/ml), asparagine (0.6 mg/ml), and HEPES buffer (2 mM). For thecontinuous exposure test each compound is added to the above mixture.Cells are placed in 35 mm petri dishes in a top layer of agar over anunderlayer of agar to prevent growth of fibroblasts. Three plates areprepared for each data point. The plates are placed in a 37° C.incubator, and are removed on day 14 for counting of the number ofcolonies in each plate. The number of colonies (defined as 50 cells)formed in the 3 compound treated plates is compared to the number ofcolonies formed in the 3 control plates, and the percent coloniessurviving at the concentration of compound can be estimated. Threepositive control plates are used to determine survival rate. Orthosodiumvanadate at 200 μg/ml is used as the positive control. If there is <30%colonies in the positive control when compared to the untreated control,the test is evaluated.

Activity of Carbendazim Against Human Tumor Colony Forming Units 1 HourExposure 1 Hour Exposure Concentration-μg/ml Concentration-μg/ml TumorType 0.5 5.0 50 0.5 5.0 10 50 Brain — — — 0/2 0/2 — 2/2 Breast 0/1 0/10/1 0/3 0/4 0/1 2/3 Cervix — — — 0/1 0/1 — 1/1 Colon 0/1 0/1 0/1 0/5 1/5— 2/5 Head & Neck 0/1 0/1 0/1 0/1 0/1 — 1/1 Kidney 0/1 0/1 0/1 0/2 0/2 —½ Lung, non-small 0/1 0/1 0/1 0/6 2/6 0/1 2/5 cell Melanoma 0/2 0/2 1/20/3 1/3 — 2/3 Mesothelioma — — — 0/1 0/1 0/1 — Ovary 0/3 0/3 0/3 0/1 2/13 — 10/13 Sarcoma 0/1 0/1 1/1 0/1 0/1 — 1/1 Stomach — — — 0/1 0/1 —1/1 Uterus — — — 0/3 0/3 — 2/3 Unknown — — — 0/1 0/1 — 0/1 Primary 0/110/11 2/11 0/4  6/44 0/3 27/41 0% 0% 18% 0% 14% 0% 66%

(4-thiazolyl)-1H-benzimidazole shows efficacy in the Human Tumor ColonyForming Units test described above when tested using continuous exposureof the cells to the (4-thiazolyl)-1H-benzimidazole.

The following table summarizes these results showing positive results ona number of cancer types:

1 Hour Exposure Continuous Exposure Concentration - μg/mlConcentration - μg/ml Compund 0.5 5.0 50.0 0.5 5.0 50.02-(methoxycarbonyl- 0/1 0/1 0/1 0/14 3/14 5/14 amino) benzimidazole(4-thiazolyl)-1H- 0/3 0/3 0/3 1/10 2/10 7/10 benzimidazole

In Vivo Studies of Carbendazim on Cancer Cell Lines

The dose response effect of carbendazim in mice infected with variouscancer types was studied in standard screening tests.

The results of these studies conducted to study the dosage response ofcarbendazim on various cancer types are summarized below. These resultsare representative of a number of tests in which a knownchemotherapeutic agent is used as the control so that the efficacy ofthe carbendazim can be compared to it. Efficacy of carbendazim has beenshown in the following cancers:

In a Prostate cancer model doses of 4000, 5000 and 6000 (mg/kg givenonce weekly) were as effective as Mitoxantrone at 70 days. It was betterthan Cytoxan at these doses of 4000, 5000 and 6000 (mg/kg given twiceweekly) through 40 days. The tables show tumor weight in mg for eachdose. The carbendazim was given once a week (p.o) and the Mitoxantronewas given by i.v. (q.d.x5).

dose day 1 day 5 day 9 day 12 day 16 day 19 peanut oil 61.6 88.9 146.8184.9 278.1 305.8 control 6000 mg/kg 62.1 92.2 140.4 162 226.5 275.99carbendazim 5000 mg/kg 63.1 100.1 116.8 138.3 280.4 246.9 carbendazim4000 mg/kg 63.1 97.5 159 192.7 282.1 311.6 carbendazim Mitoxantrone 61.991.5 120.4 150.9 169.7 187.1 1.5 mg/kg dose day 23 day 26 day 30 day 33day 37 day 40 peanut oil 385.6 592.4 518 625.2. 537.6 594 control 6000mg/kg 301.9 400.9 416.6 447.3 546.3 514.4 carbendazim 5000 mg/kg 281.3374.6 370.6 428.6 406.4 391.6 carbendazim 4000 mg/kg 316.6 368.1 351.3410.7 506.8 484.9 carbendazim Mitoxantrone 208.5 248 247.3 296.9 363 4651.5 mg/kg dose day 44 day 47 day 51 day 53 day 59 day 61 peanut oil714.1 777.4 665.7 764.8 981.3 936 control 6000 mg/kg 505.2 484 438.3499.8 492.2 480.1 carbendazim 5000 mg/kg 445.7 454.7 505.9 543.3 628.6579.1 carbendazim 4000 mg/kg 481.5 511.5 543.1 552.9 507.8 560.3carbendazim Mitoxantrone 545.6 474.9 495.8 566.2 656.8 657.4 1.5 mg/kgdose day 65 day 68 day 72 peanut oil — — — control 6000 mg/kg 581.9525.9 667.3 carbendazim 5000 mg/kg 562.3 562.3 602 carbendazim 4000mg/kg 631.2 697.8 739.1 carbendazim Mitoxantrone 775.1 820.8 707 1.5mg/kg

Both the Cytoxan and the carbendazim were given p.o. twice a week

dose day 1 day 5 day 8 day 12 day 15 day 19 peanut oil 66.9 118.2 185.8250.2 264.5 351 control 6000 mg/kg 66.7 97.3 143 193.5 237.9 316.2carbendazim 5000 mg/kg 67.9 84.9 126.8 152.8 184.2 199.5 carbendazim4000 mg/kg 67.2 110.4 157.6 192.6 238.9 298.7 carbendazim Cytoxan 66.798.4 179.4 234.6 259.7 278.1 300 mg/kg dose day 22 day 26 day 29 day 33day 36 day 39 peanut oil 416.1 446.2 555.3 802.7 868.4 1032.3 control6000 mg/kg 331.5 371.7 421.7 517.2 529.9 595.2 carbendazim 5000 mg/kg236.8 247.6 293.6 351.5 409.8 497.8 carbendazim 4000 mg/kg 330.5 347.9346.6 421.1 464.9 517 carbendazim Cytoxan 351.2 467.8 583.8 786.1 904.21143.5 300 mg/kg

In the Colon—HT29 mouse model carbendazim at doses of 4000, 5000 and6000 (mg/kg given twice weekly) it is better than Cytoxan in this model.At a dose of 3000 (mg/kg given twice weekly) it is also better thanCytoxan.

The carbendazim and Cytoxan were given twice weekly.

dose day 1 day 5 day 8 day 12 day 16 day 19 peanut oil 53.5 67.1 98.6154.6 187 236.3 control Cytoxan 51.3 60.3 42 58.5 64 87.8 300 mg/kg 6000mg/kg 53.5 55.8 45.2 62.8 46.4 59.4 carbendazim 5000 mg/kg 53.5 58 58104.3 83.2 109.7 carbendazim 4000 mg/kg 51.3 63.8 59.7 85.8 81.1 119.4carbendazim dose day 23 day 26 day 29 day 37 day 40 peanut oil 335.5433.1 499.9 786.3 984.9 control Cytoxan 137.7 174 252 407.3 503 300mg/kg 6000 mg/kg 77 78.4 60 51.3 62.5 carbendazim 5000 mg/kg 129.2 149.4133.3 185.6 185.1 carbendazim 4000 mg/kg 142.1 159.6 156.2 184.6 212.9carbendazim dose day 1 day 6 day 9 day 13 day 16 day 20 control- no 77.1172.3 231.4 348.1 409.3 478.4 treatment peanut oil 75.8 172.2 218.2300.3 344 460 control Cytoxan 76.6 132.5 152.5 142.8 188.1 226 300 mg/kg3000 mg/kg 75.8 108.1 110.4 141.4 152.3 121.7 carbendazim dose day 23day 26 day 29 control- no 582.8 710.2 867.3 treatment peanut oil 540.8701.5 863 control Cytoxan 372.7 375 478 300 mg/kg 3000 mg/kg 141.8 173.5209.5 carbendazim

In the Breast—MX-1 model, carbendazim at 4000, 5000, and 6000 (mg/kg,twice weekly) was dose responsive in slowing the growth of the tumor andwas better than Cytoxan. It is also very effective in MCF-7L breastline. At a dose of 3000, (mg/kg given twice weekly) it was equivalent toNavelbine.

The Cytoxan and the carbendazim were given twice weekly, p.o.

dose day 1 day 5 day 8 day 12 day 15 peanut oil control 70.9 208.6 5261153.6 2267.9 Cytoxan 300 mg/kg 70 32.9 4.2 3.2 0 6000 mg/kg carbendazim70.4 151.8 259.9 492.9 663.6 5000 mg/kg carbendazim 70.1 157.4 272.1535.9 856.4 4000 mg/kg carbendazim 70.3 158 320.4 626.2 1126.5

When the tumor was shrunk with Taxol first and then the carbendazimtherapy started when the tumor began to grow again on day 130, thecarbendazim treatment was begun and the tumor subsequently shrunk tozero. The Navelbine was given 1.6 mg./kg, qdx5, i.p; the Taxol was given16 mg/kg, qdx5, i.p; and the carbendazim was dosed twice weekly, p.o.The carbendazim treatment was better than Navelbine and as good as Taxolin this study.

dose day 1 day 5 day 8 day 12 day 15 peanut oil 72.9 91.4 95.2 117.1121.4 control 3000 mg/kg 70.8 98.7 90.8 110.1 106.8 carbendazimNavelbine 68.1 95.3 83.2 106.1 116.3 Taxol 68.9 85.2 52.1 36.3 27.5 doseday 20 day 23 day 26 day 29 day 35 day 37 peanut oil 157.6 171.9 170.1211.6 226.1 229.4 control 3000 mg/kg 112.5 137.4 127.9 135.2 140.1 137.9carbendazim Navelbine 135.3 160.5 157.9 162.8 195.8 212.2 Taxol 28.531.8 32.4 34.2 36.3 32.9 dose day 41 day 44 day 48 day 51 day 55 day 58peanut oil 253.2 257.7 252.1 247.8 263.9 278.3 control 3000 mg/kg 129.2128 134.6 111.9 98.7 107.9 carbendazim Navelbine 223.1 217.4 237.3 222.3255.8 251.6 Taxol 29.9 38.7 29.7 33.3 27.1 35.2 dose day 62 day 65 day69 day 72 day 76 day 79 peanut oil 274.4 252.8 275.2 277.9 274.7 296.4control 3000 mg/kg 102.3 103.7 85.1 81.4 75.2 66.1 carbendazim Navelbine254.2 257.3 306.1 301.9 307.6 340.7 Taxol 35.2 35.2 22.2 33.2 32.9 35.2dose day 82 day 85 day 89 day 93 day 97 day 100 peanut oil 277.2 276.1168.7 197.2 278.8 264.6 control 3000 mg/kg 66.3 61.9 18.1 39.3 53.9 54.2carbendazim Navelbine 322 352.1 249.9 314.9 375.6 368.6 Taxol 35.2 39.217 36.8 43.7 46.8 dose day 103 day 105 day 110 day 113 day 116 day 119peanut oil 266.1 263.1 277.2 278.3 288.9 305.3 control 3000 mg/kg 49.752.4 52.4 49.9 51.3 43.7 carbendazim Navelbine 399.3 391.1 418.8 440.6544.4 491.9 Taxol 53 53.2 50.5 48.4 65.9 65.8 dose day 124 day 131 day134 day 137 day 140 day 144 peanut oil 331 371.2 396.7 440.5 449.5 482.8carbendazim 3000 mg/kg 43.7 49.2 47.1 53.9 49.4 52.2 carbendazimNavelbine 514.9 607.3 741.5 692.6 687.4 772.3 Taxol 76.6 86.8 92.8 97.992.6 92.4 dose day 147 day 152 day 155 day 158 day 161 day 165 peanutoil 506.6 540.7 — control 3000 mg/kg 50.6 53.9 49.4 40.7 49.4 49.4carbendazim Navelbine 811 809.4 — Taxol 104.6 105.9 116 68 73.1 68 doseday 172 day 175 day 179 day 182 day 186 day 189 peanut oil control 3000mg/kg 61.2 64.6 69.6 66.3 74.1 81.3 carbendazim Navelbine Taxol 68 71.973.1 68.1 67.1 68.1 dose day 193 day 196 day 200 day 207 day 210 day 214peanut oil control 3000 mg/kg 78.6 92.2 96.7 117.3 126.2 137 carbendazimNavelbine Taxol 66.3 63.9 58.5 51.4 54.8 34.1 dose day 217 day 221 day224 day 228 day 232 peanut oil control 3000 mg/kg 142.1 162.3 167.3175.8 209.3 carbendazim Navelbine Taxol 27.5 20.8 20.8 20.8 0 dose day236 day 239 day 245 day 249 peanut oil control 3000 mg/kg 145.7 136.3158.7 197.1 carbendazim Navelbine Taxol 0 0 0 0

In a third study carbendazim was tested in mice MX-1 model with the micereceiving estrogen tablets. Estrogen accelerates the growth of thebreast cancer. At doses of 2000, 4000 and 6000 (mg/kg given once weekly)it was better than both Navelbine (1.6 mg/kg, qdx5, i.p.) and Taxol (16mg/kg, qdx5, i.p.) in this faster growing MX-1 cancer.

dose day 1 day 5 day 9 day 12 day 16 peanut oil control 109.2 307.7947.6 1702.8 3359.8 6000 mg/kg carbendazim 109.5 292.7 593.1 1279.61261.6 4000 mg/kg carbendazim 109.5 337.1 664.2 1143.3 1501.5 3000 mg/kgcarbendazim 110.4 312.4 603.6 1068 1502.8 2000 mg/kg carbendazim 110.4342.3 752.3 1447.6 1609.1 Navelbine 110.2 278.6 874.5 1528.5 2746.8Taxol 110.4 292.7 484.9 876.6 1941.5

In the Pancreas (Mia-PaCa) model, carbendazim at 3000 and 4000 (mg/kg,twice weekly) is as good as or better than gemcitabine. At 2000 mg/kgthe carbendazim was not as effective after 21 days. Gemcitabine wasgiven on days 1,4,7 and 10 i.p.; the carbendazim was given p.o. twiceweekly.

dose day 1 day 5 day 9 day 12 day 16 peanut oil 63.1 118.5 186.6 228.4294.6 control 4000 mg/kg 64 78.9 121.3 113.4 133.4 carbendazim 3000mg/kg 63.1 71.8 100.1 100.4 139.6 carbendazim 2000 mg/kg 63.7 85.2 128.4155.1 213.4 carbendazim Gemcitibine- 63.9 71.7 81.7 77.1 94.9 80 mg/kgdose day 19 day 23 day 26 day 30 day 33 day 37 peanut oil 325.9 462.8489.5 546.6 control 4000 mg/kg 119.4 157 154.2 124.3 131.4 129.2carbendazim 3000 mg/kg 131.9 146.6 131.9 140.1 135.1 110.6 carbendazim2000 mg/kg 182.2 185 189.9 214.1 206.7 217.6 carbendazim Gemcitibine-111.3 167.1 204 258.7 330.3 404.3 80 mg/kg dose day 40 day 44 day 47 day51 day 54 day 61 peanut oil control 4000 mg/kg carbendazim 3000 mg/kg105 105 120.1 124.1 124.1 118.6 carbendazim 2000 mg/kg 214.55 214.5210.9 222.4 225.6 214.1 carbendazim Gemcitibine- 503.7 80 mg/kg dose day65 day 68 day 72 day 75 day 79 peanut oil control 4000 mg/kg carbendazim3000 mg/kg 130 130 69.2 52.5 75.8 carbendazim 2000 mg/kg 217.3 217.3202.6 178.2 169.7 carbendazim Gemcitibine- 80 mg/kg

In the Panc-01 model for pancreatic cancer carbendazim at a dose of 5000mg/kg was better than Gemcitabine at 32 days. The Gemcitabine is giveni.p., q3dx4 and the carbendazim is given p.o. twice weekly to the end.

dose day 1 day 4 day 8 day 11 day 18 day 22 control- no 64.1 110.8 201.5339.7 726 1001.4 treatment peanut oil 64.3 123.8 200.3 306 740.3 1174.1control Gemcitabine 64 106.3 171.7 248.5 561.4 943.6 80 mg/kg 5000 mg/kg64.4 115.5 166.8 247 417.9 574.3 carbendazim dose day 25 day 29 day 32control- no 1183 treatment peanut oil 1126.7 control Gemcitabine 10531183 80 mg/kg 5000 mg/kg 695.8 845 807.9 carbendazim

In the Neuroblastoma (SK-N-MC) model a decrease in tumor growth at5000(mg/kg) is shown and there is favorable activity early in the studycompared to topotecan. It is expected, based on learning from the otherstudies, that a lower dose would be effective. The topotecan is giveni.p. qdx5 and the carbendazim is given p/.o. twice weekly.

dose day 1 day 5 day 9 day 12 day 16 day 19 peanut oil 454.8 1034.11533.1 1141.8 1402 1668.2 control 5000 mg/kg 474.4 505.8 799.3 841.8 7321424.3 carbendazim Topotecan 504.9 209.8 57.5 71.8 144.7 294.6 3 mg/kgdose day 23 day 25 day 31 peanut oil 1852.8 2025 control 5000 mg/kg1480.5 936 carbendazim Topotecan 648.1 886.6 1150.2 3 mg/kg

In the Rhabdomysarcoma model, a childhood cancer, at day 29 carbendazimat doses of 3000, 4000 and 5000 (mg/kg given once weekly, p.o.) is quiteeffective against this tumor model.

dose day 1 day 5 day 8 day 12 day 15 peanut oil 58.1 121.3 177 223 340.9control 5000 mg/kg 58.1 95.1 87 88.1 106.4 carbendazim 4000 mg/kg 59.5110.7 124.2 121.6 155.3 carbendazim 3000 mg/kg 58.2 110.4 136.6 176.7248.2 carbendazim Topotecan 58.3 82 53.3 24.1 26.6 3 mg/kg dose day 19day 22 day 26 day 29 day 35 day 37 peanut oil 558 689.3 894 948.3 1038.21098.5 control 4000 mg/kg 112.6 107.9 137.5 174.3 238.8 317.4carbendazim 3000 mg/kg 113 127 140.8 144.3 131.9 152.4 carbendazim 2000mg/kg 313.1 291.2 384.3 417.4 591.2 492.9 carbendazim Topotecan 13.418.9 40.9 64.2 107.9 158.9 3 mg/kg dose day 40 day 43 day 47 peanut oilcontrol 4000 mg/kg 313.9 423 171.5 carbendazim 3000 mg/kg 142.6 195.5235.8 carbendazim 2000 mg/kg 555.6 682.1 854.3 carbendazim Topotecan184.4 279.2 351.9 3 mg/kg

In the Lung cancer model MV522 all groups received Taxol and thenreceived treatments shown starting 10 days later. At doses of 2000,3000, 4000 and 5000 (mg/kg given twice weekly, p.o.) tumor growth wassuppressed in a dose response and the tumor was shrunk in the 5000 mg/kggroup. Day 1 is the start of the treatment with carbendazim.

dose day 1 day 4 day 8 day 11 day 15 day 18 Control - no 6.6 5.6 7.318.3 39.6 80.2 treatment peanut oil 6.7 16.9 29.7 50.6 84.9 140.6control 5000 mg/kg 6.5 7.1 16.1 12.7 18.8 27.2 carbendazim 4000 mg/kg6.6 8.5 17.6 16.2 20.7 26.6 carbendazim 3000 mg/kg 6.5 7.1 11.9 9.2 14.116 carbendazim 2000 mg/kg 6.5 10.3 19.9 33.8 44.8 40.5 carbendazim doseday 22 day 25 day 29 day 32 day 36 control - no 128.8 180.1 270.7 307.3594.6 treatment peanut oil 233.9 341.4 488.7 control 5000 mg/kg 21.531.4 40.5 29.5 54 carbendazim 4000 mg/kg 40.8 39.7 58.9 47.3 62.5carbendazim 3000 mg/kg 19.9 23.4 37.5 28.9 44.5 carbendazim 2000 mg/kg37.9 43.5 49.8 41.1 53 carbendazim dose day 39 day 42 day 46 day 49 day52 day 56 control - no 572.4 670.1 treatment peanut oil control 5000mg/kg 63 86.8 86 1 0.5 carbendazim 4000 mg/kg 62.5 32 carbendazim 3000mg/kg 59.2 69.3 72.6 160.7 207.1 267.4 carbendazim 2000 mg/kg 42.9 47.160.3 107.4 107.4 175.3 carbendazim dose day 59 day 63 day 66 control -no treatment peanut oil control 5000 mg/kg carbendazim 4000 mg/kgcarbendazim 3000 mg/kg 75.4 77 94.5 carbendazim 2000 mg/kg 214.9 193.7188.9 carbendazim

In a Medulloblastoma model (IMR32) a dosage dependent effect is seenwith carbendazim at doses of 5000, 4000, and 3000 (mg/kg, p.o. twiceweekly). It is compared with Topotecan (i.p. qdx5).

dose day 1 day 7 day 9 day 12 day 15 day 19 peanut oil control 57.6 82.1103.8 107.5 143.1 182.7 5000 mg/kg 57.2 74.9 86.4 94.6 102 125.4carbendazim 4000 mg/kg 58.1 89.3 108 112.9 152.9 162.3 carbendazim 3000mg/kg 58.1 73.8 99.3 105.1 119.7 153.4 carbendazim Topotecan 3 mg/kg57.8 22.6 23.6 15.7 3.6

In the Murine Melanoma—B16 mice model doses of 4000, 5000 and 6000(mg/kg) were at least equal in tumor suppression compared to cytoxan at30-60 days. Since this tumor is a liquid tumor, there is no change intumor weight. The results are summarized in Table 1 below.

In the P-388 model for leukemia there was a dose responsive effect andthe results were good. See Tables 2 and 3.

The data in Tables 1, 2 and 3 are reported in T/C which is interpretedusing the following scale:

T/C < 125 no activity T/C = 125-150 weak activity T/C = 150-200 modestactivity T/C = 200-300 high activity T/C = 300 with long term survivorsexcellent activity

long term survivors for P388 is >30 days, for B16 it is >60 days. TheNCI Measure of Success is T/C=125.

Use of Carbendazim in the Treatment of B 16

This study was performed in black mice injected i.p. with B-16 mousemelanoma which many researchers believe is the most predictive model forefficacy in humans. Carbendazim was equally effective as Cytoxan in thismodel at treating B-16. The activity is dose responsive. Death is theend point in this model.

TABLE 1 Dosage (mg/kg) twice weekly T/C weight change (%) PositiveControl 5000 198 −0.64 Cytoxan at 300 mg/kg 2500 169 +3.16 one dose 2000169 −11.63 T/C 191, −8.43% 1250 124 +1.99 weight change 1000 176 −4.75 500 141 −0.64

TABLE 2 In a repeat test the following results were achieved: Dosage(mg/kg) twice weekly T/C weight change (%) Positive Control 6000 183−0.38 Cytoxan at 300 mg/kg 5000 167 −1.44 one dose 4000 138 +3.86 T/C161, −4.28% weight change

TABLE 3 Use of Carbendazim in the treatment of P388 Dosage (mg(kg) T/Cweight change (%) Positive Control 4000 189 −9 cytoxan at 125 mg/kg 2000148 +8 is curative; weight 1000 129 +15 change −14

Accordingly, carbendazim retards the growth of both solid and liquidtumors in vivo.

These studies confirm previous in vivo studies in which carbendazim wastested in in vivo models for different types of cancer.

Additional in vivo cancer studies are presented in a tabular form below.The data is presented in the following format—dose regimen inparenthesis, dosage in mg/kg: tumor growth suppression (tg supp.), toxicdeaths/number of test subjects (deaths), % weight change (wt).

TABLE 4 Cancer Positive Control dose tg supp. deaths wt. dose tg supp.deaths wt. MX1 xenograft- (twice weekly) Cytoxan (1 dose) 6000 70% 1/9−4 300 complete  0/10 −7 shrinkage 5000 63%  1/10 −4 NCI Measure ofSuccess: 58% 4000 48%  0/10 −1 MCF-7L (breast) (twice weekly) Cytoxan (1dose) 6000 10/10 300 100%  3/10 −15 5000 100%   7/10 −8 NCI Measure ofSuccess: 58% 4000 94%  5/10 −7 DU-145 (twice weekly) Cytoxan (1 dose)6000 33%  1/10 −6 300  0%  0/10 +6 5000 48%,  1/10 −8 NCI Measure ofSuccess: 58% 4000 52%  0/10 −5 A549 (lung) Cytoxan 2500 10/10 125  69% 0/10 −1  500 57%  0/10 +10 HT29 (colon) (twice weekly) Cytoxan (1 dose)6000 97%  9/10 −25 300  45%  0/10 +10 5000 79%  2/10 −8 NCI Measure ofSuccess: 58% 4000 78%  3/10 −12 3000 65%  2/10 −6 2000 36%  0/10 SK-MES(twice weekly) Taxol (5 doses) 6000 69%  3/10 −2  20  75%  0/10 +1 500044%  1/10 +2 Navelbine (5 doses) 4000 45%  2/10 +3  2 26%  1/10 −5 NCIMeasure of Success: 58%

Carbendazim demonstrated the ability to reduce tumor growth in micemodels for breast, lung, colon, murine melanoma and leukemia. The dataare summarized in Table 5 and Table 6 below.

TABLE 5 Positive Tumor Control Tumor Growth (Dosage in Growth CancerDosage Decrease (%) mg/kg) Decrease MXI-breast  500 mg/kg 42% cytoxanregression 2500 mg/kg 37% (125) A549 - lung  500 mg/kg* 57% cytoxan 69%(125) HT29 - colon 2500 mg/kg** 54% cis-plat (4) 59% *in this model 2500mg/kg was toxic; **in this model 500 mg had no effect

In the same test 2-(4-thiazolyl)-1H-benzimidazole showed no activityagainst MXI breast cancer tumors implanted subcutaneously under the miceskin.

TABLE 6 Increased Positive Cancer Dosage Life Span Control P388-luekemia1000 mg/kg 129% cytoxan curative (125) 2000 mg/kg 148% 4000 mg/kgdecreased life span −9% B16 melanoma 1000 mg/kg 131 % navelbineincreased life (2) span - 265% 2000 mg/kg 163% 4000 mg/kg 187%

In the same test 2-(4-thiazolyl)-1H-benzimidazole showed no activityagainst P388.

In the same test 2-(4-thiazolyl)-1H-benzimidazole showed no activityagainst B 16.

The in vivo and in vitro data support the assertion that carbendazim hasbroad scale efficacy against multiple cancer types.

Initial efficacy of carbendazim appears to be comparable to the bestavailable drugs used for the treatment of any particular cancer type.Furthermore, with continuous treatment, breast cancers do not come backas usually happens with cytoxan and taxol in breast cancer orgemcitabine in pancreatic cancer.

It is equally effective against p53 deficient/defective cell lines,unlike most existing cancer treatment drugs.

It is excellent in B16 mouse melanoma, which is believed by many peopleto be the best predictive model for efficacy in humans.

These same benzimidazole derivatives are effective against virusesincluding HIV, influenza, rhinoviruses and herpes viruses. Thebenzimidazole derivatives can be used alone or in combination with otherfungicides.

The following examples illustrate the effectiveness of thiabendazole,2-(4-thiazolyl)-1H-benzimidazole, against HIV and the benzimidazolesderivatives against a number of viruses.

The results of these HIV studies are summarized in more detail below:

Thiabendazole is effective at totally suppressing virus production inchronically infected cells. The extra cellular viral count goeseffectively to zero or non-detectable levels. Thiabendazole does notkill the chronically infected cells though it does reduce the rate ofcell proliferation at active concentrations. Thiabendazole does notaffect CD₄ expression in uninfected cells. At effective concentrationsthiabendazole slows but does not alter the normal cellular RNA orprotein synthesis of either infected or non-infected cells.Thiabendazole is effective in a variety of chronically infected celltypes (this effect is not cell type specific.)

Thiabendazole is effective against a variety of HIV virus strains. (Notvirus strain specific—although some variance by strain is observed;SK-1>IIIB>RF) Also thiabendazole is not effective on SIV in vitro or invivo.

After 20 months no resistant virus strains to thiabendazole havedeveloped in tests designed to do so. Resistance develops in six monthsor less in this test for existing HIV drugs with resistance strains forprotease inhibitors developing in about 3-4 months.

Thiabendazole does not adversely affect the activity of existing HIVdrugs, AZT, 3TC, ddC, ddl or protease inhibitors (saquinavir andindinavir) in acutely infected cells, nor do any of these existing drugsinterfere with the efficacy of thiabendazole in chronically infectedcells. It is used in combination with these drugs. Thiabendazole is alsoeffective against protease inhibitor resistant viruses.

Thiabendazole confers temporary suppression of viral production from 4to 80 days after treatment stops. This is unique and a useful featurewhenever one has problems with compliance.

The results of these studies are summarized in detail below:

HIV Virus Replication Study

Thiabendazole was tested in chronically infected HIV virus. These cellpopulations contain integrated copies of the HIV genome andconstituitively produce HIV at relatively high levels (CEM-SK1, U937-SK1and H9-SK1 from Frederick Research Center, Maryland) or are latentlyinfected and only produce virus after stimulation with phorbol esters,tumor necrosis factor or IL6 (U1 and ACH2). Virus production was reducedin all cell lines tested and thiabendazole did not stimulate virusproduction from the latently infected cells. Reductions in virusproduction were observed when quantifying supernatant reversetranscriptase activity, supernatant p24 as well as intracellular p24,indicating the compound inhibits virus production at a step ofreplication prior to production of intracellular proteins.

Quantification of the infectivity of virions produced from the infectedcells demonstrates reductions in the number of infectious virions inparallel with reductions in supernatant RT or p24, indicating thecompound reduces the amount of virus produced, but not the quality ofthe virions. Inhibition of virus production from the chronicallyinfected cells was observed at concentrations which were nontoxic to thetarget class. Thiabendazole inhibited virus production at concentrationsgreater than 1-10 μg/ml.

Toxicity to the chronically infected cells was similar to that observedwith the uninfected cells. Evaluation of thiabendazole on chronicallyinfected cells was performed by evaluation of thymidine (DNA), uridine(RNA) and leucine (protein) incorporation into cellular macromolecules.Inhibition of cellular macromolecule synthesis paralleled the toxicityof the compound as would be expected and did not occur at lower nontoxicconcentrations found to inhibit virus production from the chronicallyinfected cells.

After 28 days of treatment on chronically infected cells, the toxicityof the compound to the target cells appeared similar in both uninfectedand chronically infected cells. The compound does not preferentiallykill HIV-infected cells. Reductions in the level of virus productionwere stable and were observed at concentration greater than 10 μg/ml forthiabendazole.

These results suggest thiabendazole can quickly reduce the level ofvirus production from cell populations chronically infected with HIV-1and the antiviral effect is maintained with prolonged compound exposure.This reduction of virus production occurs at concentrations which arenontoxic to the host cell and which have no effect on the synthesis ofcellular DNA, RNA and protein.

Virus Resistance Studies

Chronically infected HIV cells were cultured in the presence ofthiabendazole at 1 μg/ml for the first month, 5 μg/ml for the secondmonth, 10 μg/ml for the third month, 20 and 40 μg/ml for the fourthmonth and 80 μg/ml for the fifth and sixth months. At the end of eachmonth, the cells were evaluated for virus production compared tochronically infected cells not treated with the compound. For each ofthe six months of treatment experience, no change in the antiviraleffect of the compound was noticed and the toxicity of the compoundremains identical. Thiabendazole remains active against HIV and thatresistance was not rapidly achieved via the selection of resistantviruses or adaptation of the cells to prevent compound induced toxicity.Virus production remains totally suppressed from cultures treated withthiabendazole at 40 and 80 μg/ml.

The present invention includes a method of treatment HIV with thebenzimidazole compounds without inducing formation of benzimidazolederivative or thiabendazole resistant HIV.

Reappearance of Virus Production from Chronically Infected CellsPreviously Treated

Chronically infected cells which were treated with compound forprolonged periods were washed free of compound and cultured to determineif, and when, virus production would resume. Cultures in which treatmentresulted in the total elimination of virus production were used in theseassays. These cultures included chronically infected cells cultured inthe presence of 20, 40, and 80 μg/ml of thiabendazole. Within 4 daysvirus production resumed from the cells cultured in the presence of thelower concentrations of thiabendazole (20 μg/ml and 4 μg/ml). Virusproduction resumed at the 40 μg/ml concentration of thiabendazole by day12. At the highest concentrations virus production was observed atapproximately day 70.

The present invention includes a method of treatment HIV with thebenzimidazole derivative or thiabendazole and delaying the reappearanceof HIV in plasma following initial treatment of HIV with an antiviralagent or thiabendazole.

Infectability of Cells Treated with Thiabendazole

Cells which were pretreated with thiabendazole for a long period of timewere washed free of compound and used as a target cell population. Thecells were split into 3 populations and labeled Group 1, 2 or 3. Group 1was treated with the compound for 24 hours (at the same concentrationused in the prolonged treatment phase), washed free of compound andcultured in the presence of infectious virus and fresh compound. Group 2was pretreated for 24 hours, washed free of compound and cultured in thepresence of infectious virus only. Group 3 was cultured for both thepretreatment and the infection phases in fresh medium only (no virus orcompound). Virus production from the cell populations was identicalirrespective of the culture conditions. These results indicate that thechronically infected cells treated for prolonged periods were notsuper-infected with HIV.

Additional Chronic HIV studies

Chronic HIV-1 infected cells U1 were derived from an acute HIV-1infection of the promonocytic cell line, U937. The chronic HIV-1infected cells, ACH-2 were derived from an acute HIV-1 infection of theT cell line, A3.01.

These cells were cultured in medium and the phorbol ester, PMA. PMAcauses the cells (both U1 and ACH-2) to be activated and not divide butit also causes the U-1 cells to differentiate. This results in fewercells in the PMA-treated cultures than the media alone cultures. Cellviability was measured when these cell lines were treated with the testcompound.

Both cell lines constituitively produce a small amount of HIV-1. ACH-2cell lines tend to produce more HIV-1 than U1 cells as shown by p-24ELISA. When either cell line is cultured in the presence of PMA there isan increase in the quantity of HIV-1 produced as measured by the p-24antigen ELISA.

In addition, the number of institute positive HIV mRNA expressing cellsper microscopic field is measured. Comparisons can be made from thesenumbers since the same number of cells were adhered to the glass slidesfor each drug concentration (10×10⁶cells/ml).

These cells were treated with test samples. Thiabendazole at 60 μg/mlsuppressed replication in the HIV monocytes by 74% and the T-cell HIVreplication was increased by 26%. The positive control was interferonwhich suppressed HIV monocytes replication by 80%. AZT showed noactivity in this model.

2-(Methoxycarbonylamino)benzimidazole suppressed replication in the HIVmonocytes by 9% and the T-cell HIV replication was increased by 44%. Thepositive control was interferon which suppressed HIV monocytesreplication by 80% and suppressed T-cell HIV replication by 60%.

Acute HIV Testing

In an in vitro acute model for HIV 2-(methoxycarbonylamino)benzimidazoleinhibited viral replication by 100% at 4 μg/ml and AZT inhibited viralreplication by 98% at 1 μg/ml. 2-(4-thiazolyl)-1H-benzimidazoleinhibited viral replication by 98% at 60 μg/ml.

The therapeutic index (TI), the ratio of the toxic dose of drug toefficacious dose of drug for 2-(4-thiazolyl)-1H-benzimidazole is 2.8versus 12, 500 for AZT. The TI for 2-(methoxycarbonylamino)benzimidazoleis 1.8.

In vivo Herpes

In an in vivo herpes screening test of 2-(4-thiazolyl)-1H-benzimidazoleat a dose of 200 mg/kg dose, 10% of the mice survived with a 10.4 meandeath date; at 100 mg/kg dose 50% of the mice survived with a 9.2 meandeath date. The positive control was acyclovir at 75 mg/kg dose; 60% ofthe mice survived with a mean death date of 17.2 days. In the same test2-(methoxycarbonylamino)benzimidazole showed no activity.

Other Tests

Both 2-(4-thiazolyl)-1H-benzimidazole and2-(methoxycarbonylamino)benzimidazole were tested in an in vitroinfluenza model and showed no activity.

In an in vivo model for influenza 2-(4-thiazolyl)-1H-benzimidazole wastested at 200 mg/kg, 67% of the mice survived with a mean death date of8 days; at 100 mg/kg, 62% survived with a mean death date of 8.7 days.The positive control was amantadine (75 mg/kg) with 100% of the micesurviving for 21 days. 2-(Methoxycarbonylamino) benzimidazole was notactive in the same test.

Both 2-(4-thiazolyl)-1H-benzimidazole and2-(methoxycarbonylamino)benzimidazole were tested in an in vitro herpesmodel and showed no activity.

Both 2-(4-thiazolyl)-1H-benzimidazole and2-(methoxycarbonylamino)benzimidazole were tested in an in vitrorhinovirus model and compared to A-36683. The therapeutic index (TI),the ratio of the toxic dose of drug to efficacious dose of drug, for2-(4-thiazolyl)-1H-benzimidazole is 1-2 and for2-(methoxycarbonylamino)benzimidazole is 1-3 versus 1000-3200 forA-36683.

The demonstrated effectiveness of the compounds of the present inventionin the human breast and lung tumor xenograft models indicate that thecompounds of the present invention are useful for the treatment of solidtumors in man, and, in particular, tumors of the breast and lung. Thisconclusion is further supported by published analyses correlatingpre-clinical test results with clinical efficacy of anti-cancer agents.For example, see: Goldin and Venditti (1980) Recent Results CancerResearch 76: 176-191; Goldin et al. (1981) Eur. J. Cancer 17: 129-142;Mattern et al. (1988) Cancer and Metastasis Review 7: 263-284; Jacksonet al. (1990) Cancer Investigations 8: 39-47. Based on these publishedanalyses, the exceptional high level of antitumor activity exhibited bythe presently claimed compounds provide strong evidence that thecompounds claimed in present invention have therapeutic utility in thetreatment of cancer in man and that they will improve the quality oflife of the patient.

What is claimed is:
 1. A method for treating breast cancer in a patientin need thereof, the method comprising administering to a patient atherapeutically effective amount of a composition comprising abenzimidazole compound of the formula:

wherein, X is hydrogen, halogen, alkyl of less than 7 carbon atoms, oralkoxy of less than 7 carbon atoms; n is a positive integer of less than4; Y is hydrogen, chloro, nitro, methyl, ethyl, or oxychloro; R ishydrogen, an alkyl group of from 1 to 8 carbon atoms, oralkylaminocarbonyl wherein the alkyl group has from 3 to 6 carbon atoms;and R₂ is NHCOOR₁, wherein R₁ is an aliphatic hydrocarbon of less than 7carbon atoms; or a pharmaceutically acceptable salt thereof, or mixturesthereof, wherein the breast cancer had survived treatment with anotheranticancer agent prior to treatment with the benzimidazole.
 2. Themethod of claim 1 wherein said benzimidazole compound has the formula:

wherein R is hydrogen; and R₂ is NHCOOR₁, wherein R₁ is methyl, ethyl orisopropyl.
 3. The method of claim 1 wherein said pharmaceuticallyacceptable salt is a hydrochloride salt.
 4. The method of claim 1wherein said benzimidazole compound is in the form of a parenteralcomposition suitable for administration by injection.
 5. A method fortreating breast cancer in a patient in need thereof, the methodcomprising administering to the patient a therapeutically effectiveamount of a composition comprising 2-(methoxycarbonylamino)benzimidazolewherein the breast cancer had survived treatment with another anticanceragent prior to treatment with the benzimidazole.
 6. The method of claim5 wherein said 2-(methoxycarbonylamino)benzimidazole is in the form of apharmaceutically acceptable salt thereof.
 7. The method of claim 6wherein said pharmaceutically acceptable salt is a hydrochloride salt.